Reprogramming Human Astrocytes Into PV Inhibitory Neurons

The pathogenesis of epilepsy is very complex.Currently,it is widely believed that the abnormality of excitability and inhibitory dynamic Balance(E/I Balance)mediated by inhibitory interneurons is the main cause of epilepsy,and targeting inhibitory intern eurons may be a very effective strategy for the treatment of epilepsy.The reprogramming system can convert human astrocytes into different types of neurons,including dopaminergic neurons and glutamatergic neurons.However,the efficient reprogramming of human astrocytes into interneurons,especially those expressing parvalbumin(PV),has not yet been reported.In order to obtain PV interneurons in vitro,we first initiated reprogramming of human primary astrocytes by overexpression of reprogramming factor OCT4 and knockout of cell cycle regulator p53.But we found that the reprogramming efficiency was very low.In order to further improve the reprogramming efficiency of astrocytes,15 small molecules involved in pluripotent stem cell differentiation,direct transdifferentiation of somatic cells,and directed differentiation of interneurons were selected as candidate compound library.Four small molecule drugs,CHIR99021,SB431542,Repsox and Y27632,can promote the conversion efficiency of MAP2 positive neurons.Further testing on different combinations,we found that the combination of CHIR99021,SB431542,Repsox and Y27632(CSBRY)could significantly promote the reprogramming efficiency of human astrocytes.Surprisingly transcriptome sequences analysis of CSBRY treatment on day 14,we found that CSBRY cocktail significantly activated the expression of NKX2.1,SP8,SOX6 and other Medial ganglionic eminence,MGE specific expression gene.Immunostaining results showed that 26%of cells expressed NKX2.1.Secondly,in order to further improve the efficiency of astrocyte reprogramming to interneurons,we examined different concentrations of Shh signaling pathways agonist SAG,Wnt signaling pathways activator CHIR99021,FGF8 and BMP4 after CSBRY treatment,the results showed that 500 nM SAG can significantly promote the astrocytes into MGE progenitor cells,and reprogramming efficiency reached to 60%.Finally,we found that 5μM DAPT treatment for 5 days further promoted the induction of MGE cells into 34%PV+interneurons,and also increased the number of neurite branches and total neurite length,which indicated accelerating maturation of PV+ interneurons.Therefore,this study established a novel method for efficient reprogram ming of PV interneurons from human astrocytes,which is expected to develop a new strategy for the intervention of epilepsy based on PV interneurons.